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TISSUE-SPECIFIC STEM CELLS |
aNational Research Laboratory for Cardiovascular Stem Cell, Seoul National University College of Medicine, Seoul, Korea;
bDepartment of Internal Medicine, Seoul National University Hospital, Seoul, Korea
Key Words. Angiogenesis • Endothelial progenitor cells • Ischemia • Cell signaling • Cell adhesion molecules • Gene therapy
Correspondence: Hyo-Soo Kim, M.D., Department of Internal Medicine, Seoul National University Hospital, 28 Yongon-dong Chongno-gu, Seoul 110-744, Korea. Telephone: 82-2-2072-2226; Fax: 82-2-766-8904; e-mail: hyosoo{at}snu.ac.kr
Received on June 24, 2006;
accepted for publication on March 26, 2007.
First published online in STEM CELLS EXPRESS April 5, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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On the other hand, Akt is a key downstream effector molecule of VEGF and SDF-1 in endothelial cells [10, 11] and increases endothelial survival [12, 13], nitric oxide (NO) production [14], migration [15], permeability [10], and expression of adhesion molecules (e.g., ICAM-1) [16]. Therefore, the function of Akt in a ischemic organ might be essential for EPC homing. In this study, we demonstrated that phosphorylated Akt (phospho-Akt) is upregulated in ischemic muscle and is important in regulating the entrapment of EPCs, and thus is a candidate target to improve the efficacy of therapeutic angiogenesis in a murine hind limb ischemia model.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Mouse Bone Marrow Harvesting, EPC Culture, and Characterization of EPCs
All procedures were approved by the Institutional Review Board of Seoul National University and were performed in accordance with the Institutional Animal Care and Use Committee of Seoul National University Hospital. Six-week-old C57BL/6J mice were anesthetized with 100 mg/kg ketamine + 10 mg/kg xylazine i.p. Both tibial and femoral bones were removed, the ends of the bones were cut, and bone marrow was irrigated with phosphate-buffered saline (PBS). Irrigates were fractionated with Histopaque-1083, and mononuclear layers were harvested and washed three times with PBS.
To transplant ex vivo-expanded EPCs, bone marrow mononuclear cells (BMMNC) were cultured on gelatin-coated six-well plates with EGM-2 BulletKit medium (Clonetics, San Diego, http://www.cambrex.com) for 7 days. We characterized the cultured cells with immunofluorescence and flow cytometry using antibodies against Sca-1, c-kit (eBiosciences, San Diego, http://www.ebioscience.com), CD34 (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), flk-1 (Chemicon, Temecula, CA, http://www.chemicon.com), Tie-2 (eBiosciences), von Willebrand factor (vWF) (DAKO, Glostrup, Denmark, http://www.dako.com), CD45 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), CD11b (eBiosciences), and F4/80 (eBiosciences), as well as BS-1 lectin binding (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI)-acetylated low density lipoprotein (acLDL) uptake (Invitrogen, Carlsbad, CA, http://www.invitrogen.com).
Mouse Hind Limb Ischemia Model
Six-week-old C57BL/6J mice (Biogenomics, Seoul, Korea, http://www.orient.co.kr) were used for all animal experiments. Six-week-old nude mice (originated from the BALB/c strain; Biogenomics), however, were used for evaluation of EPC-induced neovascularization combined with Akt gene transfer to maximize the difference of neovascularization and avoid immunologic rejection of transplanted cells, because nude mice have a limited spontaneous neovascularization and immunodeficiency.
To induce muscle ischemia, mice were anesthetized as described above, and a unilateral femoral artery was ligated and then removed as previously described [17]. Daily observation and wound dressing were done to avoid significant infection of the wounds.
EPC Injection
One day after unilateral femoral artery excision, 1 x 106 cultivated EPCs in 100 µl of endothelial basal medium (EBM)-2 medium without growth factors were administered systemically by cardiac puncture using an insulin syringe with a 27-gauge needle as previously reported [18]. EPCs were labeled with a fluorescent dye, Vibrant-DiI (Invitrogen), for tracking and quantification as explained below.
Tissue Preparation and Immunofluorescence
Mice were sacrificed at predetermined time points by administration of an overdose of sodium pentobarbital. For immunofluorescent staining, the calf muscles were rinsed in PBS to remove excess blood, snap-frozen in liquid nitrogen, and stored at –80°C.
Ten-micrometer-thick histological sections were prepared from snap-frozen tissue samples. Immunofluorescent staining was performed using goat anti-VEGF-A antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), anti-phospho-Akt antibody (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), anti-SDF-1 antibody, anti-ICAM-1 antibody, anti-vascular cell adhesion molecule 1 (VCAM-1) antibody (R&D Systems), or fluorescein isothiocyanate-conjugated BS-1 lectin (Sigma-Aldrich). We observed the specimens using a confocal microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) or an inverted fluorescent microscope (Olympus, Tokyo, http://www.olympusglobal.com).
Network Formation on Matrigel
Cultured EPCs (1 x 105) were plated on growth factor-reduced Matrigel (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) and then incubated in EGM-2 medium at 37°C for 2 weeks. Representative fields were taken by an inverted microscope.
NO Production by VEGF Stimulation
Intracellular NO formation was analyzed using diamino-fluorescein-2 diacetate (DAF-2 DA) (Dai-ichi Pharm. Co., Ltd., Tokyo, http://www.daiichipharm.co.jp), a membrane NO-specific fluorescence indicator, as performed previously [18]. Briefly, EPCs were gently washed twice with Ca2+-free PBS and then bathed in Krebs-Henseleit buffer containing L-arginine (1 mM) and DAF-2 DA (10 µM) for 15 minutes. VEGF (50 ng/ml) was added to the wells, and then the cells were incubated at 37°C for an additional 20 minutes.
Western Blot of Akt, ICAM-1, and VCAM-1
To investigate Akt signaling in endothelial cells, we performed immunoblotting of endothelial cells stimulated with VEGF and SDF-1. Either VEGF or SDF-1 was added to human umbilical vein endothelial cells (HUVEC). Cells were harvested 2 hours later for immunoblot of phospho-Akt, total Akt, ICAM-1, and VCAM-1. Protein extracts were obtained from cell pellets. Protein was separated on a 12% polyacrylamide gel and then electroblotted onto nitrocellulose membranes. Membranes were then blocked with 5% nonfat dried milk in 0.2% Tween phosphate-buffered saline (T-PBS) and probed with 1:1,000 rabbit polyclonal anti-phospho-Akt or total Akt antibody (Cell Signaling Technology) or with rabbit polyclonal anti-ICAM-1 or VCAM-1 antibody (Santa Cruz Biotechnology) overnight at 4°C. After incubation with primary antibody, blots were washed three times in T-PBS and incubated for 1 hour with 1:2,000 anti-rabbit horseradish peroxidase IgG (Santa Cruz Biotechnology).
Adenovirus-Mediated Gene Transfer of Akt
To modulate Akt activity, we introduced adenoviral vectors to either endothelial cells in vitro or nonischemic and ischemic muscle in vivo. For endothelial cells in vitro, a multiplicity of infection (m.o.i.) of 50 of either adenoviral vector (ad-) expressing myrAkt (ad-myrAkt) or ad-DNAkt (a kind gift from Kenneth Walsh, amplified and used in our laboratory [19]) was given. Incubation time was 24 hours before further experiments. For muscle tissue in vivo, 1 x 108 plaque-forming units (pfu) were directly injected. ad-green fluorescent protein (ad-GFP) or ad-ß-galactosidase (ad-ßgal) was used as control.
Quantification of EPC Entrapment
To quantify the EPC homing to the ischemic organ, we used two methods, as we reported previously [8]. Briefly, one method was direct counting of DiI-labeled EPCs in the histologic section of the muscle; these EPCs were harvested 15 hours after transplantation. The other was transplanting radiolabeled EPCs and then measuring specific radioactivity per gram of muscle tissue using a gamma counter after correcting for radioactive decay. Before cellular transplantation, EPCs in suspension were washed with PBS and incubated with DiI at a concentration of 2.5 µg/ml in serum-free basal medium for 10 minutes at 37°C or with technetium-99m hexamethyl propyleneamine oxime (Tc-99m-HMPAO) at a concentration of 1 mCi per 1 x 107 EPCs for 30 minutes. After PBS washes, the cells were resuspended in EBM-2 medium.
Evaluation of Incorporation and Transendothelial Migration of EPCs
To see the effect of Akt signaling on incorporation and transendothelial migration of EPCs, 8-µm pore membrane was seeded with HUVEC after being coated with fibronectin. After 2 days, the HUVEC monolayers on membrane reached full confluence. Two types of human EPCs [18], early and late EPCs, were labeled with DiI fluorescent dye, washed three times, and resuspended with EBM at a concentration of 106 cells per milliliter. HUVEC monolayers were prestimulated with 50 m.o.i. of ad-myrAkt, ad-DNAkt, or ad-ßgal for 12 hours. Subsequently, after washing the monolayer of HUVEC, 100 µl of EPC suspension was applied on transwells in a 24-well plate. Immediately, 600 µl of EBM containing 0.1% fetal bovine serum and SDF-1 (200 ng/ml) was placed in the lower chamber. After incubation for 24 hours at 37°C in a CO2 incubator, cells in the lower chamber were counted. The cells in the lower chamber were divided into two groups, which are illustrated in Figure 1A. One group is defined as incorporation into pre-existing endothelial cells; these cells attached to lower surface of the membrane with HUVEC. The other is transendothelial migration (TEM); these cells are on the bottom of lower chamber. The pore membrane only coated with fibronectin also showed EPC migration patterns (Fig. 1B, 1C) similar to those of the membrane covered with HUVEC monolayer (Fig. 1D, 1E). However, membranes covered with fibroblast did not show any EPC migration (Fig. 1F, 1G), which indicated that transcellular migration was an endothelial cell-specific phenomenon.
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Evaluation of Fibrosis and Enhanced Healing of Hind Limb
To evaluate enhanced tissue healing of the ischemic muscle in addition to neovascularization, we performed Masson's trichrome staining to measure the fibrosis fraction in the hind limb. We also measured the circumferences of the ischemic thighs before the hind limb ischemia operation and 21 days later.
Bone Marrow Transplantation Model
The murine BM transplantation model was performed as described previously [7]. Harvested BM from GFP-transgenic mice was injected into recipient male C57BL/6J mice (n = 8; 6 weeks old). After an engraftment period of 4 weeks, hind limb ischemia was induced as described above, and then 1 x 108 pfu of ad-myrAkt, ad-DNAkt, or ad-ßgal was injected into several points of thigh muscle. Three weeks after the operation, mice received 200 µg of tetramethylrhodamine isothiocyanate-conjugated BS-1 lectin (Sigma-Aldrich) intravenously and were sacrificed 30 minutes later.
Statistical Analysis
All data are presented as mean ± SEM. Intergroup comparisons were performed by Mann-Whitney or Kruskal-Wallis nonparametric tests. Probability values of p < .05 were interpreted to denote statistical significance. SPSS version 11.0 was used for analysis (SPSS, Inc., Chicago, http://www.spss.com).
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RESULTS |
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Ischemia-Induced VEGF and SDF-1 Activated Akt Signaling, Which Led to Upregulation of Adhesion Molecules
One day after femoral artery resection, VEGF-A and SDF-1 were highly expressed in the ischemic limb (Fig. 3A, 3B) in contrast to the sham-operated nonischemic limb (Fig. 3C, 3D), as shown by immunofluorescence. VEGF-A was expressed mainly in the skeletal muscle cells and partially in endothelial cells, whereas SDF-1 was derived from stromal cells making up the interstitium.
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Phosphorylation of Akt Increased ICAM-1 Expression in Endothelial Cells in Ischemic Limb
We confirmed that ischemia-induced ICAM-1 expression in the ischemic muscle, which we reported previously [8], was modulated by Akt in vivo. ICAM-1 expressed on the vascular endothelium in ischemic muscle receiving ad-myrAkt was markedly enhanced (Fig. 3F) compared with that receiving ad-ßgal (Fig. 3G), whereas it was attenuated by ad-DNAkt (Fig. 3H). Therefore, the upregulation of phospho-Akt may be associated with the ICAM-1 overexpression during ischemia. On the other hand, VCAM-1 expression in the ischemic muscle did not show any noticeable change by treatment of adenoviral vectors modulating phosphorylation of Akt (Fig. 3I–3K).
Transendothelial Migration of EPCs Is Under the Control of Akt in Endothelial Cells
When we tested incorporation and transendothelial migration of EPCs using a modified Boyden chamber, early EPCs showed two patterns, whereas late EPCs showed just one. Some early EPCs fell to the bottom of the lower chamber after transendothelial migration (Fig. 4A–4C, photograph of early EPCs in lower chamber), whereas the others attached to the lower part of membrane with HUVEC (Fig. 4D–4F, photograph of early EPCs attaching to the lower surface of membrane). We called the former TEM and the latter incorporation, as shown in Figure 1. All late EPCs, however, attached to the lower part of membrane without falling to the lower chamber (Fig. 4G–4I, photograph of late EPCs attaching the lower surface of membrane).
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Local Akt Gene Transfer Can Induce EPC Homing Even in Normal Hind Limb
To test whether activated Akt is sufficient for induction of EPC homing and whether it directly controls EPC homing, we examined whether activated Akt alone could induce EPC homing without VEGF or SDF-1 in normal limb. To obtain phospho-Akt overexpression in normal muscle tissue, we injected ad-myrAkt intramuscularly into the normal limb. In contrast to the muscle receiving ad-ßgal (Fig. 5A–5C), overexpression of phospho-Akt was observed in endothelial cells of vessels (Fig. 5D–5F). One day after transfer of either ad-myrAkt or ad-ßgal, we transplanted ex vivo-expanded EPCs with DiI labeling for quantification. ad-myrAkt transfer increased the entrapment of EPCs (Fig. 5G) significantly more than control gene did (Fig. 5H). The number of trafficking cells was significantly higher in the ad-myrAkt-injected muscle (Fig. 5I), which suggested that Akt is a sufficient signaling pathway for EPC recruitment.
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We confirmed that initial better recruitment of EPCs led to more neovascularization in the ischemic muscle using a combination study of bone marrow transplantation from GFP-transgenic mouse and viral transgenesis with myrAkt and DNAkt. We found that GFP-positive bone marrow-derived cells were colocalized with BS-1 lectin-positive endothelial cells in the ischemic muscle on postoperative day 21 (Fig. 6C). ad-myrAkt enhanced neovascularization by bone marrow-derived cells compared with ad-ßgal, whereas ad-DNAkt reduced new vessel formation in the ischemic muscle (Fig. 6D).
The increased homing of EPCs resulted in an increased neovascularization. Capillary density on postoperative 21-day was highest in the ad-myrAkt-transferred ischemic muscle and lowest in the ad-DNAkt-transferred ischemic muscle (Fig. 6E, 6F). In addition, recovery of blood flow, which was assessed by laser Doppler perfusion analyzer, was excellent in the ad-myrAkt-transferred hind limb by day 21 (Fig. 6G). In contrast to ad-myrAkt, ad-DNAkt transfection significantly hindered the recovery of blood flow to ischemic limb.
We also confirmed that ad-myrAkt gene transfer significantly preserved muscle mass of the ischemic muscle compared with the control vector or the ad-DNAkt, which was shown by the changes of thigh circumference (Fig. 6H) and reduced tissue fibrosis of the ischemic muscle (Fig. 6I–6K).
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DISCUSSION |
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The Ex Vivo-Expanded EPCs from Mouse Bone Marrow
We used murine bone marrow-derived mononuclear cells for EPC culture in the present study. After several days of culture, they showed endothelial characteristics, such as acLDL uptake, BS-1 lectin binding, and expression of flt-1 and flk-1, as we recently reported [8]. After 14 days of culture, these cells continued to have endothelial characteristics, such as Tie-2, CD31, and vWF, as well as stem cell markers such as Sca-1, c-kit, and CD34. We confirmed the endothelial function of the cells by capillary network formation on Matrigel and NO production in response to VEGF in vitro. Some of them, however, also showed monocyte/macrophage markers, CD45, CD11b, and F4/80, which suggested that cultured cells were a heterogeneous population. The heterogeneity of the cells makes it difficult to determine which cells are functioning in the various assays conducted in vitro and in vivo in the present study because EPCs have been known to contribute to neovascularization in part by paracrine effects, in part by providing endothelial cells to new vessel [20]. In any case, the number of EPCs recruited to the neovasculogenic foci is important for the effect of cell therapy, and therefore we investigated the role of Akt in the homing of the heterogeneous EPCs.
The Multistep Process of EPC Homing
Vajkoczy et al. reported that embryonic endothelial progenitor cells were recruited to the microvasculature of tumors through arrest within the microvasculature, extravasation, forming multicellular clusters, and then incorporation into functional vascular networks [4]. We observed a similar heterogeneous phase of EPC entrapment in the ischemic muscle. Transplanted EPCs initially seemed to be attached within the vessel. Thereafter, a portion of EPCs incorporated into the vascular endothelial cells, whereas others migrated through the endothelial cells and invaded the interstitium. We do not think that this is a sequential phenomenon of monoclonal EPCs because we used heterogeneous EPCs. Although we did not observe the real-time homing process using intravital microscopy, 7-day-cultured EPCs obviously showed heterogeneous behavior in both in vitro and in vivo. Therefore, to analyze the EPC homing process, we should examine not only the initial attachment but also incorporation and transendothelial migration of EPCs into the interstitium. From this point of view, we assessed the effects of Akt on EPC homing in terms of adhesion molecules, incorporation, and transendothelial migration.
Akt as a Key Signaling Molecule in the Ischemic Muscle
VEGF and SDF-1 are known to be closely related to ischemia [21, 22], recruit EPCs in the ischemic organ [5, 6, 7, 22], and activate Akt in endothelial cells [10, 11]. In this study, we demonstrated that VEGF and SDF-1 were induced and secreted by the ischemic muscle cells or stromal cells, which led to induction of phospho-Akt in endothelial cells, suggesting that phospho-Akt is a key signaling molecule to mediate the action of VEGF and SDF-1 in ischemic limb. There are many angiogenic cytokines that are expressed in the ischemic organ [21], and some of them have been shown to recruit EPCs to the ischemic organ [6, 7]. However, Akt is the key modulator of many growth factors, such as angiopoietin-1 [23, 24] and hepatocyte growth factor [25], and drugs, such as statins [26–28]. Therefore, it would be a good choice to target Akt, which is the common pathway of these molecules, rather than to look into each molecule as an adjunctive therapy for EPC transplantation.
Akt Gene Transfer to Normal Muscle Increased the Entrapment of EPCs
Ischemia and the resulting inflammation produce various molecular events in the ischemic muscle [21, 29]. To determine the role of Akt in EPC entrapment without other confounding molecules, we introduced myrAkt gene transfer into normal limbs and systemically administered EPCs to the mice by cardiac puncture. In contrast to the control gene, myrAkt gene transfer increased the entrapment of EPCs in the normal muscle, which suggested that Akt might be a key molecule sufficient for EPC entrapment.
Low efficiency of EPC homing to old myocardial infarction or nonischemic cardiomyopathy has become the main obstacle to vascular delivery strategy of stem cells in clinical situations [30]. Adjunctive modulation of genes such as Akt seems a fascinating modality to induce new signals for stem cell homing and thus enhance the efficacy of this treatment.
The Role of ICAM-1 as a Downstream Molecule of Akt in EPC Entrapment
Recently, ICAM-1 was found to be upregulated in the ischemic muscle and to play an important role in EPC homing [8, 31, 32]. Furthermore, EPCs have ß2-integrin, the counterligand of ICAM-1, which is also known to be essential in activation and entrapment of EPC [9]. In the present study, we demonstrated that ICAM-1 could be modulated by Akt gene transfer in the ischemic muscle, suggesting that ICAM-1 might be one of the downstream molecules of Akt. Some molecules, such as nitric oxide synthase and HIF-1 [16, 31, 33], have been suggested to mediate upregulation of ICAM-1 by Akt, although details of the signaling pathway from the phosphorylation of Akt to ICAM-1 expression should be evaluated further in future studies, as should the difference between ischemic diseases and inflammatory diseases.
Akt Regulates Transendothelial Migration of EPCs
Akt is known to regulate vascular permeability through Akt-mediated phosphorylation of endothelial nitric oxide synthase, which modulates vasodilation and intraluminal surface area [14]. In addition, Akt signaling is essential for actin reorganization and directed endothelial cell migration [15]. These changes in vascular endothelial cells make the microvasculature more active and leaky. In this situation, EPCs could easily transmigrate into the interstitium and be recruited in the ischemic muscle. The results in the present study support the role of Akt in transendothelial migration of EPCs because ad-myrAkt transfer to HUVEC monolayer of the Boyden chamber increased the transendothelial migration of EPCs and, furthermore, increased tube-like formation of outgrowth ECs (late EPCs), whereas ad-DNAkt did the reverse. Akt has a role in diverse cellular processes that contribute to the angiogenesis, and Akt signaling within vascular endothelial cells in the ischemic muscle may function as an important modulator of EPC homing and neovascularization.
Akt as an Adjunctive Molecule in Cell Therapy; Cell-and-Gene Hybrid Therapy
We may treat ischemic disease by either local injection of EPCs or systemic administration of EPCs. Local injection would be better in terms of the number of EPCs delivered in a target organ. Local injection, however, requires a more invasive procedure than systemic or catheter-based administration. Therefore, systemic or catheter-based administration seems to be preferred for therapeutic angiogenesis of ischemic heart disease or ischemic limb disease. However, when we and another group evaluated the distribution of the EPCs that were introduced systemically in mice, we found that the majority of the transplanted EPCs existed in the spleen or the bone marrow [8, 34].
Therefore, we think that the mere mobilization, systemic administration, or vascular delivery of EPCs cannot guarantee their incorporation in ischemic tissues. For this reason, an adjunctive method to enhance the homing of EPCs to target tissue may be needed. Several groups have suggested some adjunctive methods to increase the efficiency of cell therapy [6, 7]. The elucidation of the molecular mechanism of the proposed methods may produce more effective therapy in the future, and here we suggest Akt as an important target molecule that stands at common signaling pathway of the angiogenic or EPC-recruiting cytokines.
In the present study, we have shown that modulation of Akt not only controlled EPC trafficking to the ischemic hind limb but also enhanced the efficacy of neovascularization and healing by cell therapy for the critical ischemia. Therefore, ad-myrAkt gene therapy would be a good choice for the pretreatment before systemic administration of EPCs to increase neovascularization. Given the accumulation of knowledge, the future direction of stem cell therapy may be a combination of more-tailored gene therapy (cell-and-gene hybrid therapy) to enhance homing and ex vivo expansion or endogenous mobilization to increase EPC number.
In conclusion, Akt plays a key role in the homing of EPCs to the ischemic muscle through increased attachment and transendothelial migration. In addition, its modulation enhances the homing of EPCs and new vessel formation in target organs. These novel findings suggest that the modulation of the homing mechanism may be used as a therapeutic strategy to improve the efficacy of stem cell therapy.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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, p < .01), whereas ad-DNAkt reduced EPC homing. (C, D): The ischemic muscles were harvested on day 21 after operation in the GFP transplantation model. Endothelial cells were stained with premortem administration tetramethylrhodamine isothiocyanate-labeled BS-1 lectin 30 minutes before harvest. Scale bar = 100 µm. (C): Arrows indicate that GFP-positive (green), bone marrow-derived cells differentiated to BS-1 lectin-positive (red) endothelial cells. (D): Double-positive new vessels were most frequently observed in the ad-myrAkt group on postop day 21. (E, F): The ischemic muscles were harvested on day 21 after operation and stained with fluorescein isothiocyanate-labeled BS-1 lectin to measure the capillary density. ad-myrAkt gene transfer increased capillary density compared with the control gene transfer. In contrast, ad-DNAkt decreased capillary density (n = 4;